Method for differentiating mesenchymal stem cells into neural cells

ABSTRACT

A method for differentiating mesenchymal stem cells of bone marrow into neural cells comprises culturing the mesenchymal stem cells in a medium containing epidermal growth factor(EGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor(HGF), and the neural cells produced thereby can be employed for the treatment of a neural disease.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for differentiatingmesenchymal stem cells in bone marrow into neural cells by culturingthem in a medium containing epidermal growth factor(EGF), basicfibroblast growth factor(bFGF) and hepatocyte growth factor(HGF), and acomposition for treating a neural disease comprising the neural cells asan active ingredient.

BACKGROUND OF THE INVENTION

[0002] Stem cells have the ability to divide indefinitely in culture andgive rise to specialized cells constituting a tissue upon stimulation bya specific differentiation stimulus.

[0003] Stem cells are divided into embryonic stem cells(ES cells) andtissue-specific stem cells depending on their differentiation potencies.ES cells are isolated from the inner cell mass(ICM) of embryos at theblastocyst stage and are pluripotent, i.e., they are capable ofdifferentiating into virtually every type of cells found in an organism.

[0004] In contrast, tissue-specific stem cells appear at a stage oforgan formation during the embryonic development and they areorgan-specific and multipotent, i.e., they are generally committed togive rise to cells constituting a specific organ. These tissue-specificstem cells remain in most of adult organs and perform the critical roleof continually replenishing the loss of cells occurring normally orpathologically. Representative tissue-specific stem cells includehematopoietic stem cells and mesenchymal stem cells present in bonemarrow. Hematopoietic stem cells give rise to various blood cells suchas erythrocytes and leukocytes; and mesenchymal stem cells, to the cellsof connective tissues, e.g., osteoblasts, chondroblasts, adipocytes andmyoblasts.

[0005] Recently, clinical applications of the stem cells have drawn anincreasing interest since the successful isolation of human embryonicstem cell. The most noticeable potential application of the stem cellsis their use as a perfect source of cell supply for a cell replacementtherapy. Hardly curable diseases, e.g., neurodegenerative disease suchas Parkinson's and Alzheimer's diseases, quadriplegia resulting fromspinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiacinfarction and liver cirrhosis, are caused by the disruption andpermanent functional disorder of the cells constituting an organ, andthe cell replacement therapy, wherein the loss of cells is replenishedfrom the outside, has been presented as an effective remedy.

[0006] However, notwithstanding the obvious benefit of the cellreplacement therapy, there exist many limitations in its clinicalapplications. Specifically, the conventional method, wherein fullydifferentiated cells isolated from the tissues of a donor aretransplanted into a patient, has the problem that it is difficult toobtain a sufficient amount of cells to be supplied to the patient. Inorder to solve this problem, cells of a specific tissue differentiatedfrom an isolated embryonic stem cell or differentiated cells fromisolated and proliferated tissue-specific stem cells can be employed ina cell replacement therapy.

[0007] Hitherto, it has been proven that mouse embryonic stem cells canbe differentiated on a culture dish into various cells such ashematopoietic cells, myocardial cells, insulin-secreting pancreaticcells and neural cells. Further, several reports have demonstrated thattransplantation of the cells differentiated from stem cells is effectivein the treatment of a disease caused by the loss of cells. For instance,synthesis of myelin in a mouse increased when myelin-synthesizingoligodendrocytes differentiated from an embryonic stem cell weretransplanted into the mouse(Brustle et al., Science, 285: 754-756,1999). Blood sugar level was regulated by transplantinginsulin-secreting cells differentiated from an embryonic stem cell intoa diabetes mouse model (Soria et al., Diabetes, 49: 157-162, 2000).Further, dyscinesia caused by spinal cord injury was remediedsignificantly by transplanting neural cells differentiated from anembryonic stem cell into a mouse having spinal cord injury(McDonald etal., Nat. Med., 5(12): 1410-1412, 1999).

[0008] However, since the human embryonic stem cell has beensuccessfully isolated only recently and there is no report on thedifferentiation of the embryonic stem cell on a culture dish into otherspecific cells than neural cells, clinical use of specific tissue cellsdifferentiated from an embryonic stem cell in a cell replacement therapystill remains on a level of possibility.

[0009] Further, since the efficiency of differentiation from anembryonic stem cell into target cells is low, there is a risk of anadverse side effect caused by other cells mixed with the target cellsduring transplantation. Accordingly, there exists a need for thedevelopment of a precise differentiation method for safer clinicalapplication of the cells differentiated from embryonic stem cells.

[0010] On the other hand, in case when tissue-specific stem cells areemployed in a cell replacement therapy, there is the problem thatlowering of the proliferating ability of the cells or differentiationinto unfavorable cells may occur during a long-term culture. Further,transplantation of neural cells is required for the treatment of aneurodegenerative disease such as Parkinson's disease. Since it isdifficult to obtain neural stem cells directly from the patient, theyare generally obtained by culturing neural stem cells isolated from thebrain tissue of dead fetus and differentiating them into neural cells.However, the use of fetal brain invites the ethical problem as well asis limited by insufficient supply, and may cause an immunologicalrejection. Further, most of neural stem cells are liable todifferentiate into astrocytes rather than neurons.

[0011] Accordingly, if it is possible to differentiate mesenchymal stemcells in patient's own bone marrow into neural cells to be used in acell replacement therapy, neural cells can be readily supplied and suchproblems as immunological rejection would not occur during treatment.

[0012] Hitherto, it has been considered that one kind of stem cellsdifferentiate only into the cells of a tissue belonging to a specificsystem. It was reported that mesenchymal stem cells formed in vitrocolonies in the presence of various growth factors such asplatelet-derived growth factor, basic fibroblast growth factor(bFGF),transforming growth factor-β (TGF-β) and epidermal growthfactor(EGF)(Kuznetsov et al., Br. J. Haematol., 97: 561, 1997; and vanden Bos C. et al., Human Cell, 10:45, 1997), and about one-third ofinitially attached cells had a multipotency, thereby differentiatinginto connective tissue cells such as osteoblasts, chondroblasts andadipocytes(Pittenger M F et al., Science, 284: 143, 1999). Further,Ferrari G. et al. reported that bone marrow is a source of myogenicprecursor cells that form new muscles(Science, 279: 1528, 1998).

[0013] Recent studies reported that mesenchymal stem cells can alsodifferentiate into the cells of the neural system. For instance,Sanchez-Ramos et al. reported that mesenchymal stem cells differentiatedinto neurons and astrocytes upon culture in the presence of retinoicacid and brain-derived neurotrophic factor(BDNF)(Exp. Neurology, 164:247-256, 2000). Dale Woodbury et al. reported that mesenchymal stemcells in bone marrow differentiated into neural cells in the presence ofantioxidants such as β-mercaptoethanol and dimethyl sulfoxide(DMSO)(J.Neuro. Res., 61: 364-370, 2000). However, the use of strongdifferentiation-inducing agents such as DMSO may cause a problem in aclinical application.

[0014] The present inventors have endeavored to discover materials thatare highly safe and capable of differentiating stem cells in bone marrowinto neural cells, and have discovered that HGF promotes thedifferentiation of mesenchymal stem cells of bone marrow into neuralcells, and also that the addition of EGF and bFGF to a culture medium,together with HGF, significantly enhances both the differentiation ofstem cells into neural cells and the amplification of the resultingneural cells.

[0015] It has been reported that EGF and bFGF stimulate thedifferentiation of neural stem cells into neurons or astrocytes whenthey are added to a serum-free medium for culturing neural stem cellsseparated from the brain tissue (Melissa et al., Exp. Neurology, 158:265-278, 1999).

[0016] HGF has been reported to enhance the viability of neurons inhippocampus and mesencephalon, and induce the growth of neurite inneocortical explant(Hamanoue M et al., J. Neurosci. Res., 43: 554-564,1996). Further, in the peripheral nervous system, it functions as anexistence factor for motoneurons(Ebens A et al., Neuron, 17: 1157-1172,1996), and involves in the growth and existence of sensory neurons andparasympathetic neurons (Fleur Davey et al., Mol. Cell Neurosci., 15:79-87, 2000).

[0017] However, it has never been reported that mesenchymal stem cellscan be differentiated into neural cells by culturing them in a mediumcontaining EGF, bFGF and HGF.

SUMMARY OF THE INVENTION

[0018] Accordingly, it is an object of the present invention to providea method for differentiating mesenchymal stem cells or mononuclear cellsin bone marrow into neural cells.

[0019] It is another object of the present invention to provide neuralcells differentiated by said method and a pharmaceutical composition fortreating a neural disease comprising the neural cells as an activeingredient.

[0020] It is a further object of the present invention to provide amethod for treating a neural disease in a mammal, which comprisesadministering the neural cells produced by the above method to a subjectin need thereof.

[0021] In accordance with one aspect of the present invention,therefore, there is provided a method for differentiating mesenchymalstem cells in bone marrow into neural cells, which comprises culturingthe mesenchymal stem cells in a medium containing epidermal growthfactor(EGF), basic fibroblast growth factor(bFGF) and hepatocyte growthfactor(HGF).

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and other objects and features of the present inventionwill become apparent from the following description of the invention,when taken in conjunction with the accompanying drawings, whichrespectively show:

[0023]FIG. 1: a photomicrograph(×100; hereinafter, same magnification isapplied) of the attached cells derived from mononuclear cells of bonemarrow cultured for 4 weeks on a medium containing 10 ng/ml EGF, 20ng/ml bFGF and 20 ng/ml HGF;

[0024]FIG. 2: a photomicrograph of the neural cells differentiated frommononuclear cells of bone marrow cultured for 8 weeks on a mediumcontaining 10 ng/ml EGF, 20 ng/ml bFGF and 20 ng/ml HGF;

[0025] FIGS. 3A and 3B: photomicrographs of a neuron and an astrocyte,respectively, which are isolated from the neural cells of FIG. 2;

[0026]FIGS. 4A to 4C: the results of immunocytochemical staining ofdifferentiated neural cells of FIG. 2, wherein FIG. 4A is NSE-positivecells; FIG. 4B, NeuN-positive cells and FIG. 4C, GFAP-positive cells;

[0027]FIGS. 5A to 5C: photomicrographs of osteoblasts, chondroblasts andadipocytes, respectively, differentiated from mesenchymal stem cells;

[0028]FIG. 6: a photomicrograph of mesenchymal stem cells takenimmediately after its inoculation on a medium containing 10 ng/ml EGF,20 ng/ml bFGF and 20 ng/ml HGF;

[0029]FIG. 7: a photomicrograph of neural cells differentiated from themesenchymal stem cells cultured for 8 weeks on a medium containing 10ng/ml EGF, 20 ng/ml bFGF and 20 ng/ml HGF; and

[0030]FIGS. 8A to 8C: the results of immunocytochemical staining of thedifferentiated neural cells of FIG. 7, wherein FIG. 8A is NSE-positivecells; FIG. 8B, NeuN-positive cells and FIG. 8C, GFAP-positive cells.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a method for differentiatingmesenchymal stem cells isolated from bone marrow into neural cells,which comprises culturing the mesenchymal stem cells in a mediumcontaining epidermal growth factor (EGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor (HGF).

[0032] Further, neural cells differentiated by said method and apharmaceutical composition for treating a neural disease comprising theneural cells as an active ingredient are also provided.

[0033] As used herein, the term “neural cells” refers to the nerve-likecells including neurons, astrocytes and microglias.

[0034] In order to differentiate mesenchymal stem cells into neuralcells, it is preferred to culture the mesenchymal stem cells in a cellculture medium containing 1 to 1,000 ng/ml, preferably, 5 to 10 ng/ml ofEGF, 1 to 1000 ng/ml, preferably, 10 to 20 ng/ml of bFGF and 1 to 1000ng/ml, preferably, 5 to 20 ng/ml of HGF for more than one week. It ismore preferable to culture the stem cells for more than 4 weeks. About 4weeks after the initiation of culture, neural cell colonies consistingof several cells are formed and, after about 8 weeks, a massive amountof neural cells are produced by the continuous growth and proliferationof the neural cell colonies.

[0035] In contrast, if the mesenchymal stem cells are cultured in thesame medium but lacking HGF, the cells can not differentiate into neuralcells. Further, if they are treated with HGF only, early differentiationoccurs, thereby preventing proliferation. This result suggests that, inthe inventive process of culturing mesenchymal stem cells to obtainneural cells, EGF and bFGF stimulate the cells to proliferate and HGFstimulates the stem cells to differentiate into neural cells. Further, asufficient amount of neural cells cannot be obtained when only one ofthe factors is employed.

[0036] Once the neural cells are completely differentiated andproliferated after 8-week culture, they can further proliferate withoutlosing their unique characteristics as neural cells even when treatedonly with EGF and bFGF. On the other hand, if the fully differentiatedand proliferated neural cells are subsequently treated with HGF only,the cells differentiate continuously without proliferation and,consequently, the number of cells decreases. Accordingly, afterculturing for 8 weeks, it is preferred to allow the neural cells toproliferate by further culturing them in a medium containing EGF andbFGF.

[0037] According to the present method using EGF, bFGF and HGF, morethan 80% of the total differentiated cells are neural cells, whichcomprises 60 to 80%, preferably 65 to 75%, of neurons and 20 to 40%,preferably 25 to 35%, of astrocytes, based on the number of cells, andcontains no microglia.

[0038] In the present invention, mesenchymal stem cells are preferablyobtained from human bone marrow. Mononuclear cells derived from bonemarrow contains hematopoietic and mesenchymal stem cells, and when theyare cultured for 1 to 2 weeks, hematopoietic stem cells easilydifferentiate into mature blood cells. Accordingly, the stem cellsproliferating thereafter are mesenchymal stem cells, which continue toprolifereate after 20 passages of subculture. The mesenchymal stem cellsdifferentiate into various connective tissue cells includingosteoblasts, chondroblasts and adipocytes.

[0039] In addition, massive production of neural cells can also beachieved when the mononuclear cells of bone marrow including themesenchymal stem cells are used in the method of the present invention,in place of the isolated mesenchymal stem cells.

[0040] The present method is advantageous especially in terms of safety,since the neural cells are differentiated from the mesenchymal stemcells by employing as inducers the specific proteins present in a humanbody, i.e., EGF, bFGF and HGF, without using a harmful differentiationinducer such as DMSO. Further, since a sufficient amount of neural cellsnecessary for the treatment of a disease can be produced from patient'sown bone marrow, a clinical application thereof would be viable owing toready availability of neural cells and reduced risk of immunologicalrejections.

[0041] The neural cells differentiated from mesenchymal stem cells inaccordance with the present invention can be used as an activeingredient of a cell composition for a cell replacement therapy for aneural disease. Non-limiting examples of neural diseases, which can betreated by using the neural cells of the present invention, includeneurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, Pick's disease, Huntington's disease, amyotrophic lateralsclerosis and ischemic brain disease. The inventive neural cells canalso be used in the treatment of various diseases caused by unusual lossof neural cells as well as dyscinesia caused by a spinal cord injury.

[0042] A cell composition for preventing or treating neural diseases canbe prepared by mixing the neural cells differentiated by the inventivemethod with a pharmaceutically acceptable excipient or carrier, or bydiluting it with a pharmaceutically acceptable diluent in accordancewith any of the conventional procedures. Examples of suitable carriers,excipients, and diluents are lactose, dextrose, sucrose, sorbitol,mannitol, xylitol, erythritol, maltitol, starches, gum acacia,alginates, gelatin, calcium phosphate, calcium silicate, cellulose,methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,water, methylhydroxybenzoates, propylhydroxy-benzoates, talc, magnesiumstearate and mineral oil. The formulations may additionally includefillers, anti-agglutinating agents, lubricating agents, wetting agents,flavors, emulsifiers, preservatives and the like. The cell compositionof the present invention may be formulated so as to provide quick,sustained or delayed release of the active ingredient after theiradministration to a mammal by employing any of the procedures well knownin the art. Thus, the formulations may be in the form of a sterileinjectable solution, suspension, emulsion, solution and the like,wherein a sterile injectable solution is preferred.

[0043] Accordingly, the present invention also provides a method fortreating a neural disease in a mammal, which comprises administering theneural cells produced by the inventive method to a subject in needthereof in an amount effective for treating the disease.

[0044] The neural cells produced by the inventive method may be injectedinto the body of a patient by any of the conventional methods. Forinstance, the method of Douglas Kondziolka(D. Kondziolka et al.,Neurology 55: 556-569, 2000) may be used. Specifically, the cranium ofthe patient is excised to create an opening having a diameter of about 1cm, and a HBSS (Hank's balanced salt solution) containing neural cellsis injected in about three spots. The injection is carried out by asyringe having a long needle and a stereotactic frame for injecting thedesired cell solution into a deep part of the brain at a correctposition. The cell composition of the present invention can beadministered via various routes including transdermal, subcutaneous,intravenous and intramuscular introduction, surgical stereotacticintroduction, intralesional introduction by vascular catheterization.

[0045] Typical unit dose of the neural cells may range from 1×10⁶ to1×10⁹ cells and they can be administered every week or every month.However, it should be understood that the amount of the activeingredient actually administered ought to be determined in light ofvarious relevant factors including the disease to be treated, theseverity of the patient's symptom, the chosen route of administration,and the age, sex and body weight of the individual patient; and,therefore, the above dose should not be intended to limit the scope ofthe invention in any way.

[0046] The following Examples are intended to further illustrate thepresent invention without limiting its scope.

[0047] Further, percentages given below for solid in solid mixture,liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/volbasis, respectively, and all the reactions were carried out at roomtemperature, unless specifically indicated otherwise.

EXAMPLE 1 Isolation of Mononuclear Cells in Bone Marrow

[0048] About 10 ml of bone marrow was taken from the pelvis of each ofhealthy volunteers and stored in glass tubes containing heparin. 30 mlof phosphate buffered saline(PBS) was added to 10 ml of bone marrow, and20 ml of the resulting mixture was slowly transferred onto 10 ml ofFicoll-Paque™ plus solution(1.077 g/ml, Amersham Pharmacia Biotech), andsubjected to density gradient centrifugation at 2000 rpm for 20 minutes.The mononuclear cell layer at the interface between the top layer andFicoll-Paque™ plus layer was recovered and subject to centrifugation at1800 rpm for 5 minutes to obtain mononuclear cells.

EXAMPLE 2 Culture of Mononuclear Cells

[0049] The mononuclear cells obtained in Example 1 were inoculated at adensity of 1×10⁶ cells/cm² to a culture flask containing a basal medium.The flask was incubated at 37° C., 5% CO₂. After 4 hours, the flask waswashed with fresh basal medium to remove non-attached cells. The basalmedium was Williams' E medium(Gibco BRL) containing 3.5 μM ofhydrocortisone(Sigma), 50 ng/ml of linoleic acid(Sigma Co.) mixed withfatty acid-free bovine serum albumin(Gibco BRL) at an equal molar ratio,0.1 μM CuSO₄.5H₂O (Sigma), 50 pM ZnSO₄.7H₂O (Sigma), 3 ng/ml H₂SeO₃(Sigma), 1.05 mg/ml NaHCO₃(Sigma Co.), 1.19 mg/ml HEPES(Sigma), 100 U/mlpenicillin(Gibco BRL), 10 mg/ml streptomycin(Gibco BRL) and 25 μg/mlamphotericin(Gibco BRL).

EXAMPLE 3 Differentiation of Mononuclear Cells into Neural Cells

[0050] In order to confirm whether the mononuclear cells obtained inExample 2 differentiate into neural cells, the mononuclear cells werecultured at 37° C., 5% CO₂ on a basal medium containing 10 ng/ml ofepidermal growth factor(Gibco BRL), 20 ng/ml of basic fibroblast growthfactor(R&D Systems) and 20 ng/ml of hepatocyte growth factor(R&DSystems) (“differentiating medium”). The differentiating medium wasreplenished two times a week.

[0051] About 4 weeks after, neural cell colonies appeared andcontinuously proliferated(see Table 1 and FIG. 1).

[0052] About 8 weeks after, neuron-form cells consisting of longprojections such as axons and short projections such as dendrites, andastrocyte-form cells consisting only with short dendrites were observed(see FIGS. 2 and 3). Further, after 8 weeks, the cells proliferated withtheir shapes unaltered, even if cultured on a basal medium containingonly EGF and bFGF(see Table 2).

[0053] However, when the mesenchymal stem cells were cultured in amedium containing only EGF and bFGF, the cells did not differentiateinto neural cells. Further, when the mesenchymal stem cells werecultured in a medium containing HGF only, the cells differentiated earlyand, accordingly, they neither grew nor proliferated(see Table 1). TABLE1 Number of cells/ml of culture after 8-week culture of mesenchymal stemcells No. of Treatment Treatment with inoculated No growth withTreatment with EGF, bFGF cells factor HGF EGF and bFGF and HGF 7.5 × 10⁷Not Not 1 × 10⁵ 2 × 10⁵ proliferated proliferated

[0054] TABLE 2 Proliferation of cells after 4- or 8-week culture ofneural cells differentiated from mesenchymal stem cells by 8-weekculture on a medium containing EGF, bFGF and HGF (No. of inoculatedcells: 1 × 10⁵) Treatment Treatment with Treatment with with EGF EGF,bFGF No growth factor HGF and bFGF and HGF After 4 Not proliferated Notproliferated 2 × 10⁵ 2 × 10⁵ weeks After 8 Not proliferated Notproliferated 5 × 10⁵ 1 × 10⁵ weeks

EXAMPLE 4 Immunocytochemistry

[0055] The neural cells obtained in Example 3, which were differentiatedfrom the mononuclear cells of bone marrow by culturing on a mediumcontaining EGF, bFGF and HGF for 8 weeks, were attached on a 1 cm² coverglass at a density of 1×10⁴ cells/cm². The cells were washed with 0.1 Mphosphate buffer for 5 minutes, fixed with 0.1 M phosphate buffercontaining 4% paraformaldehyde for 15 minutes, and washed twice with 0.1M phosphate buffered saline(PBS). The cells were treated with 0.1 M PBScontaining 1% BSA and 0.2% Triton X-100 for 5 minutes, and then, reactedfor 16 hours with first antibodies; mouse anti-human neuron-specificenolase(NSE)(Chemicon Inc.), mouse anti-human neuron-specific nuclearprotein (NeuN)(Chemicon Inc.), mouse anti-human β-tubulin III (SigmaCo.) and mouse anti-human glial fibrillary acidic protein(GFAP)(SigmaCo.).

[0056] Upon completion of the reaction with the first antibodies, theremaining antibodies were removed and the cells were washed twice with0.1 M PBS containing 0.5% BSA each for 15 minutes. A secondary antibody,rabbit anti-mouse IgG (Sigma Co.) was added thereto and incubated for 30minutes. The cells were washed with 0.1 M PBS containing 0.5% BSA eachfor 5 minutes. The reaction was carried out for 30 minutes by employingVectastain Elite ABC kit(Vector Laboratory Inc.) containingavidin-biotin. The cells were washed twice with 0.1 M phosphate buffereach for 5 minutes, DAB (3,3′-diaminobenzidine tetrahydrochloridedehydrate, Sigma Co.) was added thereto as a color developing substrate,and the mixture was allowed to react for 5 minutes. The reaction wasstopped by treating the reactants with 0.1 M phosphate buffer for 5minutes and washing them twice with the buffer each for 5 minutes. Theresulting reactants were dried and washed with distilled water for 5minutes. The cells were dehydrated and fixed by treating sequentiallywith distilled water, and 70%, 80%, 95% and 100% ethanol.

[0057] The results of the above immunocytochemical staining are shown inFIGS. 4A to 4C, wherein the differentiated cells exhibit positiveresults for neuronal markers NeuN, NSE and β-tubulin III, and astroglialmarker GFAP. These results show that the cells were differentiated intoneurons and astrocytes, as judged by their biochemical as well asmorphological characteristics. However, the cells were negative formicroglial marker OX-42, demonstrating that the mononuclear cells didnot differentiate into microglia.

[0058] The proportions of neurons(which is positive for NeuN and NSE)and astrocytes(which is positive for GFAP) in the cells differentiatedfrom the mononuclear cells of bone marrow by culturing on a mediumcontaining EGF+bFGF+HGF or EGF+bFGF for 8 weeks, were examined and shownin Table 3. TABLE 3 Negative NSE NeuN GFAP cells EGF + bFGF ab. 0.9% Ab.0.8% ab. 1.2% ab. 89% EGF + bFGF + HGF ab. 56% Ab. 75% ab. 24% ab. 20%

[0059] As can be seen from Table 3, about 80% of the totaldifferentiated cells are neural cells when cultured on a mediumcontaining EGF+bFGF+HGF for 8 weeks. The neural cells consisted of about70% neurons and about 30% astrocytes.

EXAMPLE 5 Isolation and Culture of Mesenchymal Stem Cell

[0060] In order to see whether the mesenchymal stem cells in themononuclear cells of bone marrow would differentiate into neural cells,the mononuclear cells were cultured and mesenchymal stem cells wereisolated therefrom. The mesenchymal stem cells were examined for theircapability to differentiate into various cells, as follows.

[0061] The mononuclear cells cultured as in Example 2 were inoculated ina culture flask containing DMEM (Gibco BRL) supplemented with 10% FBS(fetal bovine serum), at a density of 1×10³ cells/cm². The cells werecultured at 37° C. under an atmosphere of 5% CO₂. After 1˜2 weeks, theproliferated cells were subjected to a subculture, and the proliferationcontinued after 20 passages of subculture.

[0062] Mononuclear cells obtained from bone marrow include matureleukocytes, lymphocytes, scleroblasts, chondrocytes, muscle cells,fibroblasts, adipocytes as well as stem cells to be differentiated intothese cells, said stem cells being divided into hematopoietic andmesenchymal stem cells. The hematopoietic stem cells, which give rise toblood cells such as erythrocytes, leukocytes and lymphocytes, can notproliferate but easily differentiate into mature blood cells in ageneral culture medium. Accordingly, it can be seen that the cellsproliferating continuously as above are mesenchymal stem cells.

[0063] In order to confirm whether the proliferating cells are indeedmesenchymal stem cells, the cells were treated with various cytokinesand chemical agents and their differentiation into various connectivetissue cells, including osteoblasts, chondroblasts and adipocytes, wasexamined in accordance with the method of Pittenger et al., Science,284: 143-147, 1999.

[0064] In order to differentiate the stem cells into osteoblasts, thecells were treated with 100 mM dexamethasone, 10 mM β-glycerol phosphateand 50 nM ascorbate-2-phosphate and 10% FBS.

[0065] Further, in order to confirm the capability of the stem cells todifferentiate into chondroblasts, cultured stem cells were centrifugedto obtain cell pellets, which were treated with 100 nM dexamethasone and10 ng/ml TGF-β 3 in the absence of serum.

[0066] The differentiation of the stem cells into adipocytes was inducedby treating the stem cells with 0.5 mM 1-methyl-3-isobutylxanthine, 1 mMDexamethasone, 10 g/ml insulin, 100 nM indomethacine and 10% FBS.

[0067] Osteoblasts were examined by alkaline phosphatasestaining(Jaiswal et al., J. Cell Biochem., 64(2): 295-312, 1997);chondroblasts, by type II collagen RT-PCR(Mackay et al., Tissue Eng.,4(4): 415-428, 1998) and staining with toluidine blue; adipocytes, bystaining with oil red O.

[0068] Consequently, as can be seen from FIGS. 5A, 5B and 5C, positiveresults were observed under a light microscope in all of the samples.These results demonstrate that the mesenchymal stem cells cultured andproliferated in vitro still maintain the properties of stem cellscapable of differentiating into various connective tissue cells such asosteoblasts, chondroblasts and adipocytes.

EXAMPLE 6 Differentiation of Mesenchymal Stem Cells into Neural Cells

[0069] To examine whether the mesenchymal stem cells isolated in Example5 can differentiate into neural cells, the mesenchymal stem cells werecultured for 8 weeks in a medium supplemented with EGF, bFGF and HGF,according to the procedure of Example 3.

[0070] Consequently, like the differentiation of neural cells frommononuclear cells in bone marrow, neural cell colonies were formed after4 weeks and the neural cells grew and proliferated continuously untilthe 8th week. FIGS. 6 and 7 illustrate photomicrographs of the cellstaken immediately after inoculation and after 8-week culture,respectively.

[0071] After 8th week, the cells continuously proliferated whilemaintaining the morphological characteristics of neural cells even whenthey were treated only with EGF and bFGF.

[0072] Also, immunocytochemical staining was carried out with thedifferentiated cells, according the procedure of Example 4.

[0073] Consequently, like the results obtained for the neural cellsderived from mononuclear cells of bone marrow, the differentiated cellswere tested positive for neural markers NeuN, NSE and β-tubulin III andastroglial marker GFAP. Accordingly, it was confirmed that mesenchymalstem cells differentiate into both neurons and astrocytes(see FIGS. 8Ato 8C). FIGS. 8A to 8C show the results of immunocytochemical staining,FIG. 8A representing NSE-positive cells; FIG. 8B, NeuN-positive cells;and FIG. 8C, GFAP-positive cells.

[0074] While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method for differentiating mesenchymal stemcells into neural cells, which comprises culturing the mesenchymal stemcells in a medium containing epidermal growth factor(EGF), basicfibroblast growth factor(bFGF) and hepatocyte growth factor(HGF).
 2. Themethod of claim 1, wherein the mesenchymal stem cells are cultured in amedium containing 1 to 1000 ng/ml of EGF, 1 to 1000 ng/ml of bFGF and 1to 1000 ng/ml of HGF for more than one week.
 3. The method of claim 2,wherein the culturing is carried out for more than 4 weeks.
 4. Themethod of claim 1, wherein the mesenchymal stem cells are cultured in amedium containing EGF, bFGF and HGF for more than one week and theresulting differentiated neural cells are allowed to proliferate in amedium containing EGF and bFGF.
 5. The method of claim 1, wherein themesenchymal stem cells are isolated from human bone marrow.
 6. Themethod of claim 1, wherein mononuclear cells isolated from bone marrowand containing mesenchymal stem cells are used as a source ofmesenchymal stem cell.
 7. The method of claim 1, wherein the neuralcells contain neurons and astrocytes.
 8. A set of neural cells producedby the method of any one of claims 1 to
 7. 9. The set of neural cells ofclaim 8, which comprises 60 to 80% of neurons and 20 to 40% ofastrocytes, based on the number of cells.
 10. A cell composition fortreating a neural disease, comprising the neural cells of claim
 8. 11.The cell composition of claim 10, wherein the neural disease is aneurodegenerative disease or dyscinesia caused by a spinal cord injury.12. The cell composition of claim 11, wherein the neurodegerativedisease is selected from the group consisting of Parkinson's disease,Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophiclateral sclerosis and ischemic brain disease.
 13. A method for treatinga neural disease in a mammal, which comprises administering the neuralcells produced by the method of claim 1 to a subject in need thereof inan amount effective for treating the disease.